The goal of the proposed research is to identify small molecule inhibitors of human metapneumovirus (HMPV). HMPV is a leading cause of lower respiratory infection in children worldwide, and recurrent infections occur throughout life. HMPV is associated with severe illness in older adults, with hospitalization rates similar to influenza, and persons with underlying conditions such as asthma, immune compromise, and chronic pulmonary disease. There are no licensed vaccines or drugs for HMPV and thus there is an unmet need for antivirals. We developed a fluorescent, cell-based high-throughput screening (HTS) assay for HMPV that captures inhibitors of all stages of the viral lifecycle except budding. We optimized and validated the assay, and performed a successful pilot HTS against ~3500 small compounds in the Vanderbilt Institute of Chemical Biology (VICB) HTS facility. We propose to conduct a HTS for HMPV inhibitors using a larger but focused compound set selected from the VICB library of >150,000 compounds. Bioinformatics tools will be used in conjunction with medicinal chemistry collaborators to prescreen and exclude known toxic compounds, and to enrich for structures related to the hits discovered in our pilot screen. We plan to screen 75,000 compounds. We will perform secondary assays to confirm hits; determine IC50, CC50, and selectivity index; and define mechanisms of action of inhibitors. The results of these experiments will identify new potential antivirals for HMPV and other respiratory viruses. Further, the findings will provide a foundation for SAR work to refine compounds and conduct preclinical studies. This project offers a unique opportunity for drug discovery against an important human pathogen. The compounds discovered will also provide powerful probes of viral biology.